Investigation of the expression of the extracellular domain of human teneurin-1 in HEK cells and its prospective binding partner furin

Abstract

Teneurins are eukaryotic transmembrane glycoproteins, predominantly expressed in the central nervous system, and are molecular mediators for processes involving cell-cell interaction in neurological systems. The extracellular domains (ECDs) of teneurins have rearrangement hotspot (RHS) repeats that show sequence similarity to ABC proteins in bacteria. ABC protein complexes are a family of bacterial toxin complexes. The B-C sub-complex contains RHS repeats which form a hollow structure that shields and sequesters the C-terminal toxin peptide prior to its delivery into the host cell. Therefore, we hypothesized that the RHS repeats in the ECD of teneurin will form a similar hollow structure that contains a teneurin C-terminal associated peptide (TCAP) encoded by the carboxyl-terminal region of the teneurin protein. We also hypothesized the TCAP would be delivered to the target cell through interaction with the membrane receptor protein, furin, based on the structural similarity between B-C protein complex binding domain of the A proteins and the P domain in furins. The wider goal of our study was to use one of the human teneurin genes, teneurin-1, to investigate how its ECD is structurally organized and to use this information to shed light on the potential function of teneurins. The immediate aim of the study was to express the ECD of teneurin-1, consisting of epidermal growth factor repeats, -propeller domain, RHS repeats and TCAP using HEK 293 cells. The expression of recombinant ECD of teneurin-1 was investigated using two different strategies (transient transfection and stable transfection). However, neither strategy yielded sufficient protein for structural analysis. During the expression studies, we identified a novel interaction between the ECD of teneurin-1 and nonerythroid alpha-spectrin unexpectedly. In vitro analysis of the enzymatic activity of purified recombinant furin, a potential binding partner for teneurin, demonstrated that it was soluble and properly folded when produced in HEK 293-F cells, and suitable for interaction studies with teneurin-1. Future research is recommended to address the issues which possibly contribute to the low level of expression of teneurin-1 in HEK 293 cells. This research also forms a foundation for further research related to spectrin-teneurin interaction.